Reagents For HCV Antigen-Antibody Combination Assays

ABSTRACT

The present invention is directed to combination immunoassays, reagents and kits for simultaneous detection of HCV antigens and anti-HCV antibodies in a sample. The combination immunoassays of the present invention employ a non-ionic detergent that effectively exposes or releases the HCV core antigen from virions in a sample without interfering with the performance of other reagents such as the capture of anti-HCV antibodies by recombinant HCV antigens.

FIELD OF THE INVENTION

The present invention generally relates to immunoassays for detectionand diagnosis of HCV infection. In particular, the present inventionrelates to combination immunoassays, reagents and kits based on use of anon-ionic detergent for simultaneous detection of HCV antigens andanti-HCV antibodies in a sample.

BACKGROUND OF THE INVENTION

The hepatitis C virus (HCV), a single-stranded RNA virus, is theetiological agent of blood-borne non-A, non-B hepatitis. Chronic activeinfection with HCV often progresses to liver cirrhosis andhepatocellular carcinoma. Epidemiological studies indicate that HCVinfects more than 170 million people worldwide with a high incidence ofchronic disease ultimately progressing (more than 50% of cases) todeath. However, since it is mainly a blood-borne disease, it is possibleto identify the pathogen in blood samples and to eliminate thetransmission of disease through blood transfusion. After exposure to theHCV pathogen, there is initially no evidence of viral presence, i.e. nodetectable viral RNA or serology markers. This is referred to as the“window period” (WP). Generally, after 10 days following exposure toHCV, viral RNA can be detected while anti-HCV antibodies becomedetectable approximately 70 days later (Busch M P and Dodd R Y,Transfusion 40(10): 1157-1160, 2000). To prevent spread of HCVinfection, it is extremely important to take this observable fact intoconsideration and to establish a reliable blood-screening test, whichwould narrow the detection window. Since the commercialization of thenucleic acid amplification testing (NAT) which detects HCV RNA, thepost-transfusion HVC infection rate has been dramatically reduced. Othermethods are based on serological screening of the blood for detectingthe presence of HCV core antigen (Ortho HCV Core Antigen ELISA,Ortho-Clinical Diagnostics, Inc., Raritan, N.J.) or antibodies againstHCV polypeptides (Ortho HCV 3.0 ELISA, Ortho-Clinical Diagnostics, Inc.,Raritan, N.J.) in patient serum or plasma.

According to a survey conducted by Seme et al. (J. Clin. Virol. 32(2):92-101, 2005), the first generation HCV core antigen assay detects HCVinfection with comparable sensitivity and detection limits to thenucleic acid techniques (NAT). These assays detect HCV infection between40 to 50 days earlier than the current third generation HCV antibodyscreening assays. Although the first generation HCV core antigen assay,designed for blood screening, has significantly reduced the windowperiod, it only detects core antigen at pre-seroconversion or earlypost-seroconversion phase. Furthermore, the first generation HCV coreantigen assay is unable to detect core antigen when the antigen formsimmune-complexes with anti-core antibodies in the late seroconversionphase. Clearly, it is desirable to have a combined serology assay thatcan detect HCV core antigen in the pre-seroconversion phase as well asanti-HCV antibodies in the seroconversion phase, thus narrowing the WPsignificantly. This combined serology test can especially be a valuablemethod of blood screening in settings where the NAT test can not becarried out due to lack of equipment or competency.

Such an HCV antigen and antibody combined assay will be a significantimprovement over the current third generation serology blood screeningmethod (Ortho HCV 3.0 ELISA, Ortho-Clinical Diagnostics, Inc., Raritan,N.J.) with regards to narrowing the WP. However, one of the challengesto the successful antigen antibody combined assay is to select anappropriate detergent to disrupt HCV virions and release antigen withoutinterfering with the capture of anti-HCV antibodies by recombinant HCVantigens.

SUMMARY OF THE INVENTION

The present invention is directed to combination immunoassays, reagentsand kits for simultaneous detection of HCV antigens and anti-HCVantibodies in a sample.

The uniqueness of the combination immunoassays of the present inventionresides principally in the use of a non-ionic detergent that effectivelyexposes or releases the HCV core antigen from virions in a samplewithout any pre-processing of the sample, yet does not interfere withthe capture of anti-HCV antibodies by recombinant HCV antigens, therebypermitting simultaneous measurement of the HCV core antigen and anti-HCVantibodies.

Non-ionic detergents suitable for use in the combination assays of thepresent invention are members of the N-alkyl-N,N-dimethyl-amine oxidefamily. In a preferred embodiment, the combination assay employsLauryldimethylamine N-oxide (LDAO). In other embodiments, a derivativeor functional or chemical equivalent of LDAO is employed.

Accordingly, the present invention provides combination immunoassays invarious formats, related reagents and kits for simultaneous detection ofHCV antigens and anti-HCV antibodies in a sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to HCV antigen-antibody combinationimmunoassays, and related methods, reagents and kits.

HCV antigen-antibody combination immunoassays or “combo” assays refer toimmunoassays that simultaneously detect both HCV antigens and anti-HCVantibodies in a sample in a single assay. The antigen/antibodycombination assay methods rely on the identification and use ofantigenic and immunogenic HCV antibodies and antigens that are presentduring the early stages of HCV seroconversion, thereby increasingdetection accuracy and reducing the incidence of false results duringthe window period.

The present invention is predicated, at least in part, on theidentification of nonionic detergents that are suitable and effectivefor use in a combination immunoassay for sensitively and accuratelydetecting early HCV infection. Such detergents effectively disrupt HCVviral particles in a test sample and as a result, the released coreantigen is effectively captured and detected by monoclonal antibodyprobes. In the meantime, the detergents do not impact negatively theantibody detection either by leaching the recombinant antigens coated onthe solid-phase or by interfering with the capturing of anti-HCVantibodies during the combination assay. Therefore, the combinationassays provided by the present invention permit simultaneous detectionof both HCV antigens (including the core antigen) and anti-HCVantibodies in a sample in a single assay. Compared to the existingassays, including assays directed to detecting either HCV antigens oranti-HCV antibodies separately, the combination assays of the presentinvention circumvent the need to run two separate assays and provideimproved sensitivity and specificity for HCV detection.

In accordance with the present invention, nonionic detergents suitablefor use in a combination immunoassay include members of theN-alkyl-N-alkyl′-N-methyl-amine oxide family, which are also known aszwitter-ionic or zwitterionic detergents. The alkyl groups generallycontain not more than 15 carbon atoms each.

In one embodiment, the detergent is an N-alkyl-N-alkyl′-N-methyl-amineoxide wherein the alkyl group is a mono-chain or branched alkyl groupcontaining 9 to 13 carbon atoms, and the other alkyl group (“the alkyl”group) is a mono-chain or branched alkyl group containing 1 to 13 carbonatoms. In another embodiment, one or both the alkyl groups ofN-alkyl-N-alkyl′-N-methyl-amine oxide are substituted alkyl groups.

In a preferred embodiment, the detergent is N-alkyl-N,N-dimethyl-amineoxide characterized by the formula:

CH₃—(CH₂)n-N⁺—(CH₃)₂O⁻

wherein the length of the alkyl chain is defined by the number n, whichcan be in the range of 9 to 13, or preferably 10 to 12, more preferablyn is 11. The number “n” is at least 9; preferably at least 10; morepreferably at least 11.

In an especially preferred embodiment of the present invention, thenonionic detergent employed in an HCV combination immunoassay isLauryldimethylamine N-oxide (LDAO), having the formula ofCH₃—(CH₂)₁₁—N⁺—(CH₃)₂O⁻, which is graphically depicted as:

A non-ionic detergent can be provided to a combination assay in anyappropriate manner. For example, the detergent can be added to a testsample before the sample is placed in contact with detecting reagentssuch as capture antigens and antibodies. Alternatively, the detergentcan be mixed with a test sample simultaneously with the addition ofother reagents for detection. In a preferred embodiment, the detergentis provided in a solution (or “diluent”), which is used to dilute orsuspend a biological test sample prior to contacting the sample withcapture antigens and antibodies. Regardless of how a non-ionic detergentis provided to the assay, the concentration of the detergent in thesolution mixture when the contact with the test sample occurs should bein the range of 0.1%-2% (w/v), preferably at least 0.3% (w/v) but notmore than 1% (w/v), more preferably in about 0.5% (w/v).

Biological samples that can be tested for HCV using the combinationassays of the present invention include any sample suspected to containHCV virions, antigens or antibodies. The sample can be a biologicalfluid or tissue, including body fluids, such as whole blood, dried wholeblood, serum, plasma, or other blood components including red bloodcells, white blood cells and platelets; urine, saliva, cerebrospinalfluid, liver tissue, among others. The samples can be treated in anyappropriate manner prior to being used in the assay.

In accordance with the present invention, the anti-HCV antibodydetection part of the combination assay generally employs at least one(i.e., one or more) capture antigen that binds, therefore “captures”anti-HCV antibodies in the sample. The capture antigens are generallyantigenic peptides (containing one or more epitopes) derived from an HCVprotein encoded by the HCV genome. The sequence of the entire HCV genomeand the encoded HCV polyprotein sequence are documented in GenBank(accession #M62321 and #AAA45676, respectively) and available to thoseskilled in the art.

It should be noted that the term “HCV protein” as used herein includesboth a native, full-length protein encoded by the HCV genome (e.g., astructural or non-structural protein, or a precursor of a structural ornon-structural protein), and an artificial fusion polypeptide of twonative HCV proteins or fragments thereof (e.g., a fusion of NS3 andpartial NS4, also referred herein as “NS3/NS4”).

The term “HCV antigen” or “HCV antigens”, as used herein in reference toantigens present in a sample from an HCV-infected individual, can befull-length HCV proteins or antigenic fragments thereof.

Generally speaking, a peptide fragment of an HCV protein is antigenicand capable of binding to and capturing the respective antibodies whenthe fragment is at least 6 or 7 amino acids in length, preferably, atleast 8, or at least 9, or at least 10 amino acids; more preferably, atleast 12, 15 or 20 amino acids. Capture HCV antigens can contain morethan one epitope. The term “epitope” is well understood in the art andrefers to a molecular region or structural determinant on the surface ofan antigen capable of binding to an antibody and eliciting an immuneresponse. Capture antigens can be made recombinantly, or by conventionalchemical synthesis.

In certain embodiments, the capture antigen is an antigenic fragment ofan HCV protein selected from core antigen, E1, E2, NS2, NS3, NS4, orNS5. In a preferred embodiment, the capture antigen is derived from coreantigen, or an NS3/NS4 fusion protein as illustrated in the examplesbelow. In another embodiment, two or more peptides are used as captureantigens, which can be derived from the same or different HCV proteins.

A capture antigen can be coated on a solid phase prior to performing theassay. Alternatively, the capture antigen is conjugated with anappropriate reagent, e.g., biotin, which can mediate attachment to asolid phase after the capture antigen has formed a complex with (i.e.,has captured) anti-HCV antibodies in a liquid phase, e.g., in a cocktailELISA format.

Examples of solid phases suitable for use in the immunnassays of thepresent invention include both porous and non-porous materials, such as,for example, latex particles, magnetic particles, beads, membranes, andmicrotiter wells. The choice of solid phase material can be determinedbased upon desired assay format.

When a capture antigen is contacted with a test sample, if anti-HCVantibodies are present in the sample, such antibodies bind to thecapture antigen. The antibodies captured by the capture antigen, i.e.,the captured antibodies, can be detected using a number of approaches.

One approach utilizes a second antibody, which recognizes and binds thecaptured HCV antibodies. The second antibody, e.g., anti-human IgG, isconjugated to a signal-generating means. When the second antibody bindsto a captured (first) antibody, the signal-generating means generates ameasurable signal, which indicates the presence of the first antibody inthe test sample.

Another approach utilizes a second antigen, which can be conjugated to asignal-generating means, and is also referred to as a “detectionantigen”. Like capture antigens, a detection antigen can also containmore than one epitope, as long as one common epitope is present on boththe capture antigen and the detection antigen. Additionally, one or moredetection antigens can be used. When a detection antigen binds to acaptured antibody thereby forming a capture antigen-antibody-detectionantigen sandwich, the signal-generating means in the detection antigencan generate a measurable signal, which indicates the presence of theantibody in the test sample. This “Ag sandwich” approach is premised onthe ability of an anti-HCV antibody to simultaneously bind two identicalepitopes in two separate antigen molecules. This Ag-sandwich approachpermits highly specific detection of the anti-HCV antibodies; andbecause of this high specificity, this approach allows for the use of alarger volume of a test sample, which in turn permits a more sensitivedetection of HCV antigens present in the sample.

This signal-generating means is either itself detectable or may bereacted with one or more compounds to generate a detectable signal.Examples of signal-generating means include chromogens, radioisotopes,chemiluminescent compounds, enzymes (e.g., alkaline phosphatase, acidphosphatase, horseradish peroxidase, beta-galactosidase andribonuclease), or one partner of a binding pair (such as biotin orstrepavidin). Where an enzyme is used as a signal-generating means(e.g., alkaline phosphatase or horseradish peroxidase), addition of achromo-, fluro-, or lumo-genic substrate results in generation of adetectable signal.

In accordance with the present invention, the HCV antigen detection partof the combination assay is generally achieved using one or more pairsof a capture antibody and a conjugate antibody in an antibody sandwichformat.

The conjugate antibody is attached with any one of the signal-generatingmeans described hereinabove. The capture antibody can be coated on asolid phase, i.e., the same solid phase as the capture antigen(s)described above, prior to performing the assay. Alternatively, thecapture antibody is conjugated with an appropriate reagent, e.g.,biotin, which can mediate attachment to a solid phase after the captureantibody has formed a complex with (i.e., has captured) an HCV antigenin a liquid phase which is either already or subsequently bound with aconjugate antibody. In either approach, once a captureantibody-antigen-conjugate antibody sandwich is formed and captured ontoa solid phase, the signal-generating means can generate a measurablesignal, which indicates the presence of the antigen in the test sample.

Within each pair, there can be one or more capture antibodies eachrecognizing different epitopes, and one or more conjugate antibodieseach recognizing different epitopes. In order to permit simultaneousbinding of the capture and conjugate antibodies to the same antigenmolecule and formation of a sandwich complex, the capture antibody orantibodies should recognize different epitopes from those recognized bythe conjugate antibody or antibodies in the same pair, yet the epitopesrecognized by the capture and conjugate antibodies, respectively, shouldbe within the same HCV antigen in order to form anantibody-antigen-antibody sandwich. For example, the capture andconjugate antibodies can be directed to epitopes in any HCV protein,structural or non-structural HCV polypeptide, encoded by the HCV genome.Preferably, the antibodies are directed to epitopes within an HCVprotein selected from the core antigen, E1, E2, NS2, NS3, NS4, or NS5.In an especially preferred embodiment, the capture and conjugateantibodies in one pair are directed to, i.e., specifically bind to,epitopes within the HCV core antigen. In another embodiment, two or morepairs of capture and conjugate antibodies are used, and at least one ofthe pairs includes a capture and a conjugate antibody directed toepitopes within the HCV core antigen.

The capture and conjugate antibodies can be monoclonal antibodies orpolyclonal antibodies or combinations thereof. Preferably, monoclonalantibodies are employed.

To perform a combination assay in accordance with the present invention,one is provided with a capture antigen and either a conjugate antibodyor a detection antigen for detecting anti-HCV antibodies; a captureantibody and an HCV-specific antibody conjugate for detecting HCVantigens; and an appropriate non-ionic detergent. A test sample iscontacted with the detergent, the capture and conjugate reagents,sequentially or simultaneously, and the signal generated from theconjugate reagents can be correlated with a diagnosis of HCV infection.

In a specific embodiment, one is provided with a capture antigen, adetection antigen; a capture antibody and a conjugate antibody; and anappropriate non-ionic detergent. In a preferred embodiment, the captureantigen and the capture antibody are both coated on (covalently ornon-covalently attached to) a solid phase. A test sample is brought intocontact with the capture reagents attached to the solid phase in thepresence of the detergent. Simultaneously or preferably subsequently,the conjugate reagents (i.e., the detection antigen and conjugateantibody) are added to the reaction mixture. The signal generated as aresult of the binding of the conjugate reagents also to the solid phasecan be correlated with a diagnosis of HCV infection.

It should be noted that the capture and conjugate reagents should bechosen to avoid cross-reactivity and false-positive results. Forexample, the HCV antigens employed for detecting anti-HCV antibodies arederived from an HCV polypeptide different from the polypeptide againstwhich the capture and conjugate antibodies are directed. Alternatively,when the antigens employed for antibody detection are derived from thesame HCV protein (e.g., the core antigen) as the protein against whichthe antibody reagents are raised, the antigens used for antibodydetection should present epitopes that are not recognized by theantibody reagents used for antigen detection. Essentially, the epitopesto which antibodies bind in the antigen detection part of the assayshould not be present in the antigens used for HCV antibody detection.

The present invention also provides kits containing the various reagentsdescribed above for performing a combination assay in order tosimultaneously detect HCV antigens and anti-HCV antibodies in support ofa diagnosis of HCV infection.

EXAMPLES

The following examples describe in detail the advantages and importanceof the invention by examining the detergent Lauryldimethylamine N-oxide(LDAO) and its efficiency in achieving release of the HCV core antigenin an antigen assay and its compatibility with an HCV antibody assay.The use of detergent Lauryldimethylamine N-oxide (LDAO) in simultaneousmeasurement of HCV core antigen and HCV antibodies is also described.

It is to be understood that the examples presented hereinbelow are forillustration and are not intended to limit the scope of the presentinvention.

General Reagents for HCV Core Antigen and Anti-HCV Antibody DetectionOrtho HCV Core Antigen ELISA Test System

Ortho HCV Core Antigen ELISA (Ortho-Clinical Diagnostics, Inc., Raritan,N.J.) was used for the detection of hepatitis C nucleocapsid coreantigen (HCV core antigen) in human serum or plasma. The monoclonalantibodies coated onto the microwell solid phase were used to capturethe antigen and the secondary monoclonal antibodies conjugated to HRPwere used to detect the captured antigen. A detergent-containingspecimen diluent was used to expose the HCV core antigen for capture anddetection without a pretreatment step.

Ortho HCV 3.0 ELISA Test System

Ortho HCV 3.0 ELISA (Ortho-Clinical Diagnostics, Inc., Raritan, N.J.)was for the detection of antibody to hepatitis C virus (anti-HCV) inhuman serum or plasma. The recombinant HCV antigens c22 (amino acid2-120, core region), c200 (amino acid 1192-1931, NS3/NS4 region) and NS5(amino acid 2054-2995) coated on to the microwell solid phase were usedto capture the anti-HCV antibodies. Murine monoclonal anti-human IgGantibody conjugated to HRP was used to detect the captured humananti-HCV. This format utilizes detergent Tween 20 in the specimendiluent.

Chiron RIBA HCV 3.0 SIA

Chiron RIBA HCV 3.0 SIA (Novartis Vaccines & Diagnostics, Emeryville,Calif.) is a strip immunoblot assay used for the detection of antibodyto hepatitis C virus (anti-HCV) in human serum or plasma. The HCVantigens or peptides c22p (amino acid 10-53, core region), c33c (aminoacid 1192-1457, NS3 region), 5-1-1 and c100 peptides (amino acid1694-1735 and 1920-1935 respectively, NS4 region) and NS5 (amino acid2054-2995) were immobilized as individual bands onto test strips tocapture the anti-HCV antibodies. Murine monoclonal anti-human IgGantibody conjugated to HRP then detected the captured human anti-HCV.The assay provides additional information on antibody specificreactivity to individual antigen of an Ortho HCV 3.0 ELISA reactivespecimen.

Antigens

Two peptide derivatives were synthesized to cover the HCV core proteinsequence from amino acid 10 to amino acid 45. For HCC5N, the sequence isset forth in SEQ ID NO:1 (the N-terminus has a free amino group, asopposed to an amino group blocked by acetylation). HCC5N has tworesidues at the C-terminus, NorLeucine and Cys, which do not belong tothe HCV core protein. For HCC5-b, the sequence is set forth in SEQ IDNO:2 (the N-terminus has a free amino group). HCC5-b has two residues atthe C-terminus, Gly and Lys(Biotin), which do not belong to the HCV coreprotein. HCC5N was further conjugated to SMCC activated BSA through theC-terminal Cys of HCC5N under reducing conditions.

Recombinant HCV NS3 helicase (rNS3(h)), covering residues P1208 toT1657, was cloned and expressed in E. coli as described previously (Jin,Arch Biochem Biophys. 323(1): 47-53, 1995). A 6×His tag was fused to theN-terminus of the rNS3(h) to purify protein (resulting in SEQ ID NO:3,representing a fusion protein of the 6×His tag and rNS3(h)). A biotinconjugated rNS3(h) was made using EZ-Link Sulfo-NHS-LC-Biotin (Pierce,Cat #21335). The conjugated protein was determined to have 3 biotins perrNS3(h) molecule.

Monoclonal Anti-HCV Core Antibodies

Four monoclonal antibodies were used in the study. The production andepitope recognition sites of monoclonal antibody C11-3, C11-7 and C11-14have been described in European Patent Publication EP 0 967 484 A1. Noneof the epitope recognition sites of these monoclonal antibodies fallwithin amino acids 10-45 of HCV. The monoclonal anti-HCV antibody 12F11was raised against recombinant c22. The recognition site of 12F1 1 isaround amino acids 58-72 of HCV (core antigen). The antibodies C11-14and 12F11 were conjugated to horseradish peroxidase (HRP) using standardprocedures.

HCV Specimens

(1) anti-c22 and Anti-c33c Depleted Anti-HCV Specimens

Specimens WHO Hu-a-c33c, 41530822, 41530832 and 41530883 were originallytested anti-HCV strong reactive by Ortho HCV 3.0 ELISA and non-reactiveby Ortho HCV Core Antigen ELISA. The anti-c22 portion (anti-HCV core) ofthe antibodies was depleted by passing the specimen through an affinitycolumn in which chromatography resins were conjugated with recombinantHCV core antigen c22, the same antigen as one of the coating antigensused in the Ortho HCV 3.0 ELISA.

Specimen WHO Hu-a-c22, I-20 and 360650 were originally tested anti-HCVstrong reactive by Ortho HCV 3.0 ELISA and non-reactive by Ortho HCVCore Antigen ELISA. The anti-c33c portion (anti-partial NS3) of theantibodies was depleted by passing the specimen through an affinitycolumn in which chromatography resins were conjugated with recombinantHCV core antigen c33, the same antigen (c33c) immobilized in the ChironRIBA 3.0 SIA and a shorter version of one of the coating antigens (c200)used in the Ortho HCV 3.0 ELISA.

(2) HCV Core Antigen Reactive Specimen

HCV core antigen reactive sample Lot 16 was a plasma pool, made ofspecimens that were tested non-reactive of anti-HCV by Ortho HCV 3.0ELISA, reactive of HCV RNA by PCR, and reactive of HCV core antigen byOrtho HCV Core Antigen ELISA. HCV core antigen sample 9160834 is aplasma specimen from Boston Biomedica Inc. (BBI). The specimen wastested non-reactive to anti-HCV by Ortho HCV 3.0 ELISA, reactive to HCVRNA at about 36,379,940 IU/mL or 189,175,500 copies/mL by Versant bDNA(Bayer), and reactive to Ortho HCV Core Antigen ELISA.

(3) HCV Seroconversion Panels

HCV Seroconversion Panels tested in the present studies were allcommercially available, purchased from SeraCare Life Sciences, Inc. USA(BBI Diagnostics) and ZeptoMetrix Corporation, USA (formerImpath/BioClinical Partners, Inc.). A HCV Seroconversion Panel wascomposed of serial bleeds of human blood specimens taken from anindividual who was infected with HCV. Specimens covered a period fromanti-HCV non-reactive to reactive.

Example 1 Anti-HCV Antibody Detection (an Indirect Assay Format)

The assays were performed using reagents from an Ortho HCV 3.0 ELISAkit. 150 μL specimen diluents composed of a phosphate based buffercontaining detergent Tween-20, BSA, casein, yeast extracts and otherproteins, plus 50 μL specimen were added to each well. The plates wereincubated at 37° C. for 60 minutes, and were then washed 5 times with aPBS/Tween20-containing wash buffer. Afterwards 200 μL HRP conjugatedmurine monoclonal anti-human IgG antibody was added to each well. Theplates were incubated at 37° C. for 60 minutes and subsequently washed 6times. 200 μL OPD/substrate (one OPD tablet (Sigma, Cat #P-8287) in 6 mLof ELISA substrate buffer) was added to each well and the plates wereincubated in the dark at room temperature for 30 minutes. A 4N H₂SO₄stop solution was added at 50 μL/well. ODs were recorded at 493 nm usinga plate spectrophotometry reader. Assay cut-off setting was adapted fromHCV 3.0 ELISA that is OD of average negatives plus 0.6. The assayexceptions were:

-   -   (1) In “ELISA-1”, specimen diluent was from the kit, containing        detergent Tween 20; while in “ELISA-2”, the detergent was        replaced with N-Lauryl sarcosine (NLS), and in “ELISA-3”, the        detergent was replaced with Lauryldimethylamine N-oxide (LDAO).    -   (2) In “ELISA-1”, the HRP-conjugated murine monoclonal        anti-human IgG antibody, provided in the kit, was diluted at 1:3        prior to use; and in “ELISA-2” and “ELISA-3”, the conjugated        antibody provided in the kit was diluted at 1:2 prior to use.

As shown in Table 1, for both anti-c22 and anti-c33c depleted specimens,Tween20 (commonly used in anti-HCV antibody detection) and LDAO eachprovided better assay sensitivity, as reflected by the signal vs.cut-off ratios (“S/C”), than NLS (typically used in HCV antigendetection).

Example 2 Anti-HCV Antibody Detection (Ag Sandwich Assay Format)

In the Ag sandwich assay format, antigens labeled with biotin were usedas conjugate. The biotin-antigen conjugates bound to human anti-HCVantibodies that were captured on solid phase to form an Ag-Ab-Agsandwich. The sandwiched complexes were then detected by HRP conjugatedstreptavidin in a subsequent incubation.

COSTA™ high-binding microtiter plates were coated with 200 μL/well ofpremixed monoclonal antibodies C11-3 and C11-7 at 2.2 μg/mL of each andHCC5N-BSA at 0.1 μg/mL in 20 mM phosphate buffer (pH 7.0). The plateswere incubated at 25° C. overnight. The coating solutions were aspiratedand 200 μl/well of rNS3(h) at 2 μg/mL in PB (pH7.0) was added. Plateswere incubated at 25° C. overnight. The plates were then washed one timewith PBS/Tween wash buffer, followed by addition of 300 μL/well PBS/BSAblocking solution (1% bovine serum albumin and 30% sucrose in PBS) for 1hour at 25° C. The plates were aspirated and dried overnight at 25° C.with 10% humidity and pouched with desiccant.

Specimen diluent composition was the same as used in Example 1, exceptfor containing detergent Tween 20 in “ELISA-4”, detergent N- Laurylsarcosine (NLS) in “ELISA-5”, and detergent Lauryldimethylamine N-oxide(LDAO) in “ELISA-6”. 100 μL specimen diluent and 100 μL specimen wereadded to each well. The specimen incubation was at 37° C. for 1 hourwith shake. The plates were then washed 5 times with PBS/Tween20. 200 μLbiotin-rNS3(h) (or “rNS3(h)-b”) was added at 60 ng/mL or biotin-HCC5(HCC5-b) was added at 5 ng/mL to each well. Biotin-antigen was dilutedin conjugate diluent CD-1 (Casein Blocker in PBS, Pierce Cat #37528,supplemented with Tween 20 to 0.05% and EDTA to 2 mM). Thebiotin-antigen incubation was kept at 37° C. for 60 minutes with shake.The plates were washed 5 times and HRP-Streptavidin (JacksonImmunoResearch, Cat #016-030-084) 1:8,000 diluted in CD-1 was added at200 μL/well. The HRP-Streptavidin incubation was kept at roomtemperature for 30 minutes. The plates were then washed 6 times, afterwhich 200 μL OPD/substrate was added to each well. The plates wereincubated in the dark at room temperature for 30 minutes, and an aliquotof 50 μL 4N H₂SO₄ stop solution was added to each well. ODs wererecorded at 493 nm using a plate spectrophotometry reader. Assay cut-offwas determined as OD of negative average plus 0.300. Assay results areshown in Table 2. The results show that with the anti-c33c depletedspecimen, Tween20 and LDAO each provided better assay sensitivity thanNLS. The results also demonstrate that the antigen sandwich format(Ag-Ab-Ag) is better than the indirect format (Ag-Ab-2^(nd) Ab).

Example 3 HCV Core Antigen Detection

The HCV core antigen detection shown in Example 3 was a monoclonalantibody-HCV core antigen-monoclonal antibody sandwich ELISA. Theexposed HCV core antigen in specimen was captured by two monoclonalanti-HCV core antibodies coated on plates and detected by another twoHRP labeled monoclonal anti-HCV antibodies.

Except specimen diluent, ELISA reagents used in Example 3 were basicallyfrom the Ortho HCV Core Antigen ELISA kit. “ELISA-7” used the originalKit specimen diluent that contained 1.0% detergent N-Lauroylsarcosine(NLS). However, “ELISA-8” used specimen diluent containing 1.0%detergent Tween 20 and “ELISA-9” used specimen diluent containing 1.0%detergent Lauryldimethylamine N-oxide (LDAO).

ELISA was performed following HCV Core Antigen ELISA's protocol. 100 μLspecimen diluent and 100 μL specimen were added to each well. Thespecimen incubation was performed at 37° C. for 90 minutes with shake.The plate was washed 5 times with PBS/Tween20 and conjugate antibodieswere added at 200 μl to each well. The conjugate incubation wasperformed at 37° C. for 30 minutes. The plate was washed 6 times and 200μL OPD/substrate was added to each well. The plate was then incubated inthe dark at room temperature for 30 minutes. A 4N H₂SO₄ stop solutionwas then added at 50 μL/well. ODs were recorded at 493 nm using a platespectrophotometer reader. Assay cut-off setting was similar to HCV CoreAntigen ELISA, i.e., the OD of negative average plus 0.300.

Assay results are shown in Table 3. For specimens, NLS and LDAO eachprovided better assay sensitivity than Tween20.

Example 4 HCV Core Antigen/Anti-HCV Combination Assay

HCV core antigen/anti-HCV antibody combination ELISA was performed onthe same plates as used in Example 2. Plates were coated with monoclonalanti-HCV antibody C11-3 and C11-7, HCC5-BSA and rNS3(h). Specimendiluent was composed of 20 mM phosphate buffer (pH 7.3), 0.5M SodiumChloride, 1 mM EDTA, 1% detergent LDAO, 1% BSA, 0.03% Yeast extracts and0.01% denatured Superoxidase Dismutase (SOD), 200 μg/mL mouseimmunoglobulin G (IgG) and 0.1% 2-Chloroacetamide. The antibody/antigenconjugate was composed of HRP labeled anti-HCV monoclonal antibodyC11-14 at 4 μg/mL and HRP labeled monoclonal antibody 12F11 at 4 μg/mL,HCC5-biotin at 5 ng/mL and rNS3(h)-biotin at 60 ng/mL in a buffer of 10mM phosphate buffer (pH 7.3), 142 mM sodium chloride, 3 mM potassiumchloride, 0.1% detergent Tween 20, 1.5% BSA, 20% heat inactivatedNewborn Calf Serum, 0.03% potassium ferricyanide, 100 ug/mL mouseimmunoglobulin G (monoclonal) and 0.1% 2-Chloroacetamide. The HRPlabeled Streptavidin conjugate was composed of HRP-Streptavidin (JacksonImmunoResearch, Cat #016-030-084) 1:8,000 diluted in CD-1 (CaseinBlocker in PBS, Pierce Cat #37528, added Tween 20 to 0.05% and EDTA to 2mM).

Assay protocol was the same as described in Example 2. 100 uL specimendiluent and 100 uL specimen were added to each well. Plates were thenincubated at 37° C. for 1 hour with shake, followed by washing 5 timeswith PBS/Tween20. 200 μL antibody/antigen conjugate mixture was thenadded to each well. The antibody/antigen conjugate incubation wasperformed at 37° C. for 60 minutes with shake. The plates were thenwashed 5 times, 200 μL/well HRP-Streptavidin was subsequently added, andthe plates were incubated at room temperature for 30 minutes. Afterwardsthe plates were washed 6 times, 200 μL OPD/substrate was added to eachwell, and the plates were incubated in the dark at room temperature for30 minutes. 50 μL/well 4N H₂SO₄ stop solution was added, and ODs wererecorded at 493 nm using a plate spectrophotometry reader. Assay cut-offwas set at 0.300.

Assay results of HCV antigen/antibody combination ELISA on BBI HCVseroconversion panels are shown in Table 4. Assay results of HCVantigen/antibody combination ELISA on ZeptoMetrix HCV seroconversionpanels are shown in Table 5. It can be seen that at an early infectionstage, detection was negative in the antibody detection assay, whereasdetection was positive using the HCV RNA assay, the HCV antigen assay,or the HCV antigen-antibody combination assay. See, for example, Table4, with “PHV907”, days 4, 7 and 13 from first bleed. On the other hand,at a later stage of infection (e.g., day 164 with “PHV907”), whendetection of antigen became negative and the quantity of HCV RNA wasreduced, anti-HCV antibodies were strongly detected in the antibodydetection assay, and the S/C ratio was also well above 1 in theantigen-antibody combination assay. These results demonstrate that theHCV antigen-antibody combination assay provided a wider window ofdetection than an assay based on detection of either HCV antigen oranti-HCV antibody alone.

TABLE 1 Detergent comparison in the indirect format anti-HCV assay.ELISA assays and format ELISA-1 ELISA-2 ELISA-3 Indirect IndirectIndirect Detergent in specimen diluent Kit's conjugate dilution Tween20NLS LDAO (0.25%) (0.67%) (0.67%) 1:3 1:2 1:2 Negative plasma OD OD OD0.001 0.001 0.002 RIBA3.0 0.002 0.002 0.001 HCV Specimens Dilution c33cc22p 0.002 S/C 0.001 S/C 0.001 S/C anti-c22 depleted specimens WHO Hu-a-1:25 2+ − 2.122 3.5 0.745 1.2 1.773 2.9 c33c 1:50 2+ − 1.179 2.0 0.4140.7 0.985 1.6 1:100 1+ − 0.911 1.5 0.297 0.5 0.707 1.2 1:200 + − 0.6081.0 0.230 0.4 0.547 0.9 41530822 1:25 +/− − 1.235 2.1 0.306 0.5 1.0941.8 1:50 +/− − 0.686 1.1 0.170 0.3 0.608 1.0 1:100 +/− − 0.441 0.7 0.0890.1 0.212 0.4 41530832 1:100 2+ − 0.808 1.3 0.322 0.5 0.765 1.3 1:200 +− 0.648 1.1 0.230 0.4 0.547 0.9 1:400 +/− − 0.325 0.5 0.084 0.1 0.2010.3 41530883 1:200 + − 0.985 1.6 0.203 0.3 0.590 1.0 1:400 +/− − 0.5470.9 0.113 0.2 0.268 0.4 1:800 +/− − 0.276 0.5 0.053 0.1 0.127 0.2anti-c33c depleted specimens WHO Hu-a-c22 1:25 − 4+ 1.346 2.2 1.146 1.91.678 2.8 1:50 − 4+ 1.165 1.9 0.895 1.5 1.439 2.4 1:100 − 2+ 0.772 1.30.420 0.7 0.675 1.1 I-20 1:100 − 3+ 0.896 1.5 0.538 0.9 0.865 1.4 1:200− 2+ 0.410 0.7 0.230 0.4 0.370 0.6 360650 1:100 − 3+ 0.637 1.1 0.380 0.60.611 1.0 1:200 − 2+ 0.325 0.5 0.197 0.3 0.316 0.5 1:400 − + 0.171 0.30.108 0.2 0.173 0.3 cut-off -> 0.602 0.601 0.601

TABLE 2 Detergent comparison in the Ag sandwich format anti-HCV assay.ELISA assays and format ELISA-4 ELISA-5 ELISA-6 Ag sandwich Ag sandwichAg sandwich Detergent in specimen diluent-> Tween20 NLS LDAO (0.25%)(1%) (1%) Negative plasma Ag-biotin OD OD OD conjugate 0.044 0.012 0.013rNS3(h)-b 0.013 0.008 0.010 at 60 ng/mL RIBA3.0 0.018 0.020 0.030 HCC5-bHCV Specimens Dilution c33c c22p 0.036 S/C 0.017 S/C 0.027 S/C at 5ng/mL anti-c22 depleted specimens WHO Hu-a-c33c 1:100 1+ − 2.997 >92.997 >9 2.997 >9 rNS3(h)-b 1:200 +/− − 2.997 >9 2.997 >9 2.997 >9 at 60ng/mL 1:400 +/− − 1.567 4.8 1.922 6.4 1.446 4.8 1:800 − − 0.470 1.40.754 2.5 0.427 1.4 41530822 1:100 +/− − 0.560 1.9 1.720 5.7 0.580 1.91:200 − − 0.284 0.9 0.724 2.4 0.236 0.8 41530832 1:200 +/− − 2.997 >92.997 >9 2.997 >9 1:400 +/− − 1.418 4.7 2.538 8.5 1.984 6.6 1:800 +/− −0.232 0.8 0.720 2.4 0.360 1.2 41530883 1:200 + − 2.800 9.3 2.997 >92.997 >9 1:400 +/− − 0.510 1.7 1.560 5.2 0.838 2.8 1:800 − − 0.168 0.60.612 2.0 0.294 1.0 Cut-off -> 0.329 0.310 0.312 anti-c33c depletedspecimens WHO Hu-a-c22 1:200 − 2+ 2.119 6.5 0.174 0.5 2.997 >9 HCC5-b1:400 − 1+ 1.588 4.9 0.117 0.4 2.858 8.7 at 5 ng/mL 1:800 − +/− 1.1223.4 0.066 0.2 1.909 5.8 I-20 1:200 − 2+ 0.637 1.9 0.087 0.3 1.127 3.41:400 − 2+ 0.343 1.0 0.050 0.2 0.610 1.9 1:800 − +/− 0.185 0.6 0.046 0.10.310 0.9 360650 1:200 − 2+ 1.659 5.1 0.137 0.4 2.086 6.4 1:400 − 2+0.739 2.3 0.075 0.2 1.156 3.5 1:800 − +/− 0.337 1.0 0.056 0.2 0.592 1.8Cut-off -> 0.327 0.319 0.329

TABLE 3 Detergent comparison in HCV Core Ag assay. ELISA assays ELISA-7ELISA-8 ELISA-9 Detergent in SD NLS Tween20 LDAO (1.0%) (1.0%) (1.0%)Negative plasma OD OD OD 0.010 0.014 0.011 0.010 0.017 0.008 0.008 S/C0.013 S/C 0.009 S/C Dilution Lot 16, a pool of neat 0.722 15 0.300 5.51.852 38 plasma 1:1.5 1.075 22 0.189 3.5 1.220 25 specimens that 1:2.01.115 23 0.149 2.7 0.948 19 were all 1:3.0 0.802 16 0.103 1.9 0.621 13characterized 1:4.0 0.602 12 0.074 1.3 0.431 8.8 as anti-HCV (−), 1:5.00.490 9.9 0.064 1.2 0.335 6.8 HCV RNA (+) 1:6.0 0.379 7.7 0.051 0.90.279 5.7 and HCV core 1:8.0 0.271 5.5 0.041 0.7 0.199 4.1 Ag (+) 1:100.214 4.3 0.040 0.7 0.148 3.0 1:16 0.155 3.1 0.026 0.5 0.104 1.0 Xmillion IU/ml Dilution (copies/ml) 9160834 @ 1:5 7.28 (37.8) 1.390 280.178 3.2 1.121 23 36.4 × 10⁶ IU/ml 1:10 3.64 (18.9) 0.734 15 0.086 1.60.527 11 or 189 × 10⁶ 1:20 1.82 (9.46) 0.364 7.4 0.047 0.9 0.251 5.1copies/ml 1:30 1.21 (6.31) 0.253 5.1 0.036 0.6 0.157 3.0 1:40 0.91(4.73) 0.181 3.7 0.028 0.5 0.116 2.4 cut-off -> 0.049 0.055 0.049

TABLE 4 HCV antigen/antibody combination assay on BBI HCV seroconversionpanels anti-HCV Seroconversion Days HCV HCV Ag HCV Ag/Ab Panels from 1stRIBA 3.0 3.0 HCV RNA (HCV core) combination ID Bleed bleed c22p c33c S/Ctesting kit quantity Pos or assay From Panel Data Sheet (Neg) OD S/CPHV907 1 0 − − 0.0 HCV RNA >5 × 10⁵ pos 0.311 1.04 genotype 2 4 − − 0.0Roche >5 × 10⁵ pos 0.317 1.06 1b 3 7 − − 0.0 Amplicor >5 × 10⁵ pos 0.3711.24 4 13 1+ − 0.1 PCR (BBI) >5 × 10⁵ pos 0.437 1.46 5 18 4+ +/− 0.4copies/ml >5 × 10⁵ pos 0.877 2.92 6 21 4+ 1+ 1.0 >5 × 10⁵ pos 2.442 8.147 164 4+ 4+ 4.4  4 × 10⁴ neg >3 >10 PHV909 s1 0 − − 0.0  1 × 10⁴ pos0.305 1.02 genotype 3 2 28 1+ − 1.3  4 × 10⁴ pos 0.701 2.34 3 30 2+ −1.3  2 × 10⁴ pos 0.960 3.20 PHV910 1 0 − − 0.0 >5 × 10⁵ pos 0.599 2.00genotype 2 4 − − 0.0 >5 × 10⁵ pos 0.424 1.41 1b 3 8 3+ 1+ 3.0 >5 × 10⁵pos 1.497 4.99 4 11 4+ 3+ 6.7 >5 × 10⁵ pos >3 >10 5 15 4+ 4+ 8.0 >5 ×10⁵ pos >3 >10 PHV913 1 0 − − 0.0 >5 × 10⁵ pos 0.310 1.03 genotype 2 2 −− 0.1 >5 × 10⁵ pos 0.330 1.10 2b 3 7 2+ − 1.5 >5 × 10⁵ pos 0.867 2.89 49 2+ +/− 1.7 >5 × 10⁵ pos 1.256 4.19 PHV914 1 0 − − 0.0 >5 × 10⁵ pos0.292 0.97 genotype 2 5 − − 0.0 >5 × 10⁵ pos 0.366 1.22 2b 3 9 − −0.0 >5 × 10⁵ pos 0.307 1.02 4 12 − − 0.1 >5 × 10⁵ pos 0.325 1.08 5 16 2+− 1.2 >5 × 10⁵ pos 0.310 1.03 6 19 2+ − 2.1 >5 × 10⁵ pos 0.323 1.08 7 244+ +/− 4.5 >5 × 10⁵ pos 0.392 1.31 8 30 4+ 3+ 6.8 >5 × 10⁵ pos 1.4524.84 9 33 4+ 3+ 7.6 >5 × 10⁵ pos 2.052 6.84 PHV916 1 0 − − 0.0  3 × 10⁵pos 0.127 0.42 genotype 2 2 − − 0.0 >5 × 10⁵ pos 0.297 0.99 2b 3 7 − −0.0 >5 × 10⁵ pos 0.375 1.25 4 9 − − 0.0 >5 × 10⁵ pos 0.483 1.61 5 16 −+/− 0.3 >5 × 10⁵ pos 0.314 1.05 6 19 − +/− 1.1 >5 × 10⁵ pos 0.607 2.02 723 − 3+ 2.7  4 × 10⁵ pos 0.861 2.87 8 28 − 3+ 3.7  2 × 10⁵ neg 1.0513.50 PHV918 1 0 − − 0.0 HCV RNA >8 × 10⁵ pos 0.272 0.91 genotype 2 2 − −0.0 Roche >8 × 10⁵ pos 0.544 1.81 1a 3 7 − − 0.0 COBAS >8 × 10⁵ pos0.393 1.31 4 9 − − 0.0 Amplicor >8 × 10⁵ pos 0.660 2.20 5 14 − − 0.0 PCR(BBI) >8 × 10⁵ pos 0.441 1.47 6 16 +/− − 0.0 IU/ml >8 × 10⁵ pos 0.9983.33 7 24 3+ − 0.6 >8 × 10⁵ pos 0.368 1.23 8 27 3+ 1+ 0.8 >8 × 10⁵ pos0.627 2.09 PHV920 1 0 − − 0.0 >8 × 10⁵ pos 0.107 0.36 genotype 2 5 − −0.0 >8 × 10⁵ pos 0.891 2.97 3 7 − − 0.0 >8 × 10⁵ pos 0.304 1.01 4 13 +/−1+ 0.5 >8 × 10⁵ pos 2.292 7.64 5 16 1+ 3+ 3.1 >8 × 10⁵ pos 2.840 9.47 620 1+ 3+ 3.6  3 × 10⁴ neg 1.911 6.37 7 26 1+ 3+ >5  8 × 10⁴ neg 1.6375.46 8 28 2+ 4+ >5  2 × 10³ neg 1.533 5.11 9 33 2+ 4+ >5 BLD neg 2.3747.91 negative plasma control-1 0.016 0.05 negative plasma control-20.019 0.06 negative plasma control-3 0.011 0.04

TABLE 5 HCV antigen/antibody combination assay on ZeptoMetrix HCVseroconversion panels Seroconversion Days anti-HCV HCV Ag HCV Ag/AbPanels from 1st RIBA 3.0 HCV 3.0 HCV RNA (HCV core) combination ID Bleedbleed c22p c33c S/C testing kit quantity Pos or assay From Panel DataSheet (Neg) OD S/C 6212 1 0 − − 0.00 CHIRON 1.88 × 10⁶ pos 0.086 0.29 212 − +/− 0.15 HCV RNA 2.21 × 10⁵ neg 1.965 6.55 3 14 − +/− 0.30Copies/mL <200,000 neg 2.503 8.34 4 23 − 1+ 1.49 2.27 × 10⁵ neg >3 >10 526 − 1+ 1.87 2.03 × 10⁵ neg >3 >10 6 32 − 1+ 2.37 <200,000 neg >3 >10 737 − 1+ 2.46 3.91 × 10⁵ neg >3 >10 8 53 − 4+ 4.13 4.66 × 10⁵ neg >3 >109 55 − 4+ 4.13 3.57 × 10⁵ neg >3 >10 6215 1 0 − − 0.00 CHIRON >120 ×10⁶  pos 0.898 2.99 2 3 − − 0.01 HCV RNA  104 × 10⁶ pos 0.952 3.17 3 10− − 0.02 (bDNA)   58 × 10⁶ pos 0.456 1.52 4 20 4+ +/− 4.66 (copies/ml)  48 × 10⁶ pos 2.321 7.74 6225 10 35 − − 0.00 CHIRON <0.2 neg 0.034 0.1111 39 − − 0.00 bDNA <0.2 neg 0.021 0.07 12 45 − − 0.00 (MEq/mL) 27.02pos 0.266 0.89 13 47 − − 0.00 98.84 pos 0.678 2.26 14 52 − − 0.00 77.79pos 0.627 2.09 15 56 − − 0.01 54.71 pos 0.545 1.82 16 60 − − 0.00 >120pos 1.027 3.42 17 72 − − 0.07 9.37 pos 0.635 2.12 18 77 − 1+ 1.73 4.97pos 1.380 4.60 19 79 − 1+ 2.11 3.24 pos 1.400 4.67 6229 1 0 − − 0.0152.19 pos 0.490 1.63 2 3 − − 0.01 55.83 pos 0.528 1.76 3 7 − − 0.0155.53 pos 0.452 1.51 4 10 − − 0.01 94.76 pos 0.823 2.74 5 18 − − 0.5331.88 pos 1.346 4.49 6 21 − +/− 1.06 69.85 pos 1.636 5.45 7 25 +/− 1+1.71 51.02 pos 1.594 5.31 8 29 +/− 3+ 4.07 43.89 pos 2.590 8.63 9041 1 0− − 0.00 CHIRON <0.2 neg 0.044 0.15 2 24 − − 0.01 HCV RNA 15.78 pos0.125 0.42 3 27 − − 0.01 bDNA 2.0 63.72 pos 0.603 2.01 4 31 − − 0.01(MEq/mL), >120 pos 1.106 3.69 5 62 +/− 4+ 7.30 * one Mega 72.27 pos2.924 9.75 6 64 +/− 4+ 8.03 Equivalent/ 66.27 pos >3 >10 7 69 1+ 4+ 8.20mL 21.80 pos >3 >10 8 71 3+ 4+ 8.78 (MEq/mL) 11.24 pos >3 >10 9044 1 0 −− 0.00 is ≈ 1 105.90 pos 0.652 2.17 2 4 − − 0.01 million 52.71 pos 0.4961.65 3 17 − − 0.00 equivalents 77.18 pos 0.722 2.41 4 21 − +/− 0.69 ofHCV 71.52 pos 1.089 3.63 5 25 − 2+ 3.70 RNA. 72.82 pos 1.147 3.82 6 29 −3+ 4.51 48.83 pos 0.855 2.85 9045 1 0 − − 0.00 17.73 pos 0.361 1.20 2 2− − 0.00 16.42 pos 0.321 1.07 3 7 − − 0.00 36.19 pos 0.395 1.32 4 9 − −0.01 38.07 pos 0.557 1.86 5 26 − − 0.00 44.86 pos 0.389 1.30 6 32 − −0.04 17.78 pos 0.302 1.01 7 37 − 1+ 2.61 18.36 pos 1.314 4.38 8 41 − 2+3.51 2.32 pos 1.043 3.48 9047 1 0 − − 0.10 65.95 pos 0.474 1.58 2 2 − −0.12 81.76 pos 0.517 1.72 3 10 − − 0.09 54.85 pos 0.368 1.23 4 12 − −0.05 64.03 pos 0.501 1.67 5 19 − − 0.10 55.45 pos 0.346 1.15 6 21 − −0.09 42.97 pos 0.334 1.11 7 28 − 2+ 1.51 9.13 pos 2.921 9.74 8 30 − 4+3.92 26.58 pos 2.992 9.97 9 35 − 4+ 7.36 22.77 pos >3 >10 10 37 +/− 4+6.77 nd pos >3 >10 9054 7 52 − − 0.00 <0.2 neg 0.040 0.13 8 74 +/− −0.00 86.79 pos 0.555 1.85 9 77 1+ +/− 0.14 86.75 pos 0.532 1.77 10 82 2+1+ 3.16 59.14 pos >3 >10 9058 1 0 +/− − 0.07 90.58 pos 0.762 2.54 2 3+/− − 0.08 85.34 pos 0.764 2.55 3 7 + +/− 0.15 37.64 pos 0.487 1.62 4 101+ 1+ 0.66 48.33 pos 0.930 3.10 5 14 2+ 1+ 3.16 29.46 pos 1.209 4.03negative plasma control-1 0.016 0.05 negative plasma control-2 0.0190.06 negative plasma control-3 0.011 0.04

1. An immunoassay for simultaneously detecting HCV antigens andantibodies in a sample, comprising: providing a non-ionic detergentcomprising an N-alkyl-N,N-dimethyl-amine oxide, a first pair of acapture antigen and a detection antigen, a first pair of a captureantibody and a conjugate antibody, wherein said capture antigen and saiddetection antigen both comprise a first peptide fragment of a first HCVprotein, said capture antibody and said conjugate antibody specificallybind to a second HCV protein, and said detection antigen and saidconjugate antibody comprise one and same signal generating means;contacting said sample in the presence of said detergent with saidcapture antigen, said detection antigen, said capture antibody and saidconjugate antibody, to form a sandwich complex between said captureantigen, said detection antigen, and an anti-HCV antibody present insaid sample, and a complex between said capture antibody, said conjugateantibody, and an HCV antigen present in said sample; and measuring asignal generated from said signal-generating means as a result of theformation of said complexes, thereby simultaneously detecting HCVantigens and antibodies in said sample.
 2. The immunoassay of claim 1,wherein said N-alkyl-N,N-dimethyl-amine oxide is characterized by theformula, CH₃—(CH₂)n-N⁺—(CH₃)₂O⁻, wherein n falls in the range of 9 to13.
 3. The immunoassay of claim 2, wherein saidN-alkyl-N,N-dimethyl-amine oxide is Lauryldimethylamine N-oxide (LDAO).4. The immunoassay of claim 1, wherein said first HCV protein and secondHCV protein are independently selected from the group consisting of thecore antigen, E1, E2, NS2, NS3, NS4, and NS5.
 5. The immunoassay ofclaim 1, wherein said first HCV protein and said second HCV protein arethe same, and said capture antibody and said conjugate antibody bind toa region of said second HCV protein outside of said first peptidefragment.
 6. The immunoassay of claim 5, wherein said first HCV proteinand said second HCV protein are both the HCV core antigen.
 7. Theimmunoassay of claim 1, wherein a second pair of a capture antigen and adetection antigen is provided, wherein said capture antigen and saiddetection antigen of the second pair both comprise a second peptidefragment of an HCV protein, wherein said second peptide fragment isdifferent from said first peptide fragment.
 8. The immunoassay of claim7, wherein said first peptide fragment and said second peptide fragmentare derived from different HCV proteins.
 9. The immunoassay of claim 8,wherein at least one of said first peptide fragment or said secondpeptide fragment is a fragment of the HCV core antigen.
 10. Theimmunoassay of claim 1, wherein said capture antibody in said first paircomprises two or more antibodies.
 11. The immunoassay of claim 1,wherein a second pair of a capture antibody and a conjugate antibody isprovided, wherein said capture antibody and said conjugate antibody insaid second pair specifically bind to said second HCV protein or adifferent HCV protein.
 12. The immunoassay of claim 1, wherein saidcapture antigen and said capture antibody are attached to a solid phase.13. A kit for simultaneously detecting HCV antigens and antibodies in asample, comprising a non-ionic detergent comprising anN-alkyl-N,N-dimethyl-amine oxide, a first pair of a capture antigen anda detection antigen, a first pair of a capture antibody and a conjugateantibody, wherein said capture antigen and said detection antigencomprise a first peptide fragment of a first HCV protein, said captureantibody and said conjugate antibody specifically bind to a second HCVprotein, and said detection antigen and said conjugate antibody compriseone and same signal generating means.
 14. The kit of claim 13, whereinsaid N-alkyl-N,N-dimethyl-amine oxide is characterized by the formula,CH₃—(CH₂)n-N⁺—(CH₃)₂O⁻, wherein n falls in the range of 9 to
 13. 15. Thekit of claim 14, wherein said N-alkyl-N,N-dimethyl-amine oxide isLauryldimethylamine N-oxide (LDAO).
 16. The kit of claim 13, whereinsaid first HCV protein and said second HCV protein are independentlyselected from the group consisting of the core antigen, E1, E2, NS2,NS3, NS4, and NS5.
 17. The kit of claim 13, wherein said first HCVprotein and said second HCV protein are the same, and said captureantibody and said conjugate antibody bind to a region of said second HCVprotein outside of said first peptide fragment.
 18. The kit of claim 17,wherein said first HCV protein and said second HCV protein are both theHCV core antigen.
 19. The kit of claim 13, further comprising a secondpair of a capture antigen and a detection antigen is provided, whereinsaid capture antigen and said detection antigen of the second paircomprise a second peptide fragment of an HCV protein, wherein saidsecond peptide fragment is different from said first peptide fragment.20. The kit of claim 19, wherein said first peptide fragment and saidsecond peptide fragment are derived from different HCV proteins.
 21. Thekit of claim 20, wherein at least one of said first peptide fragment orsaid second peptide fragment is a fragment of the HCV core antigen. 22.The kit of claim 13, wherein said capture antibody in said first paircomprises two or more antibodies.
 23. The kit of claim 13, furthercomprising a second pair of a capture antibody and a conjugate antibody,wherein said capture antibody and said conjugate antibody in said secondpair specifically bind to said second HCV protein or a different HCVprotein.
 24. The kit of claim 13, wherein said capture antigen and saidcapture antibody are attached to a solid phase.